NOR‐1 Knockdown Reduces Mitochondrial Function in C2C12 Myotubes

Abstract

IntroductionSkeletal muscle is critical for whole‐body metabolic health and a prime determinant of quality of life. Mitochondrial health is compromised by several pathological states that alter muscle metabolism. Exercise is among the most effective means of preserving muscle mitochondrial health, yet rigorous exercise is often impossible in patients suffering from muscle‐related pathologies. Thus, the investigation of exercise responsive molecular targets is a requisite to identify therapeutics that improve muscle metabolic health. Neuron‐derived orphan receptor‐1 (NOR‐1) is highly responsive to exercise and is reduced in preclinical models of obesity and diabetes. The objective of the current study was to investigate the impact of NOR‐1 loss on C2C12 metabolic signaling. We hypothesized that knockdown (KD) of NOR‐1 in myotubes would perturb mitochondrial‐associated gene expression.MethodsC2C12 myotubes were transfected with control or NOR‐1 targeting siRNA to knock down NOR‐1. Protein and RNA were isolated from cells for immunoblotting and qPCR analysis, respectively. Cells were also stained with JC‐1 to evaluate alterations in mitochondrial membrane potential (MMP). NAD+ and NADH+ content was also measured in myotubes. Gene expression was normalized to ß‐actin mRNA whereas protein levels were normalized to total protein. Data were analyzed by an unpaired T‐Test with significance set at p < 0.05 and presented as means ± SEM.ResultsNOR‐1 KD cells had lower gene expression of PGC‐1α, TFAM, and MFN2 and several proteins involved in energy production were altered (ND2, CYCS, CPT‐1ß, FABP4). Myosin protein content and gene expression of MYHI, MYHIIA, and MYHIIX were also reduced. Protein abundance of mitochondrial electron transport system complexes were reduced and both BAX and DRP1 abundance were enhanced after NOR‐1 KD, which was coincident with a diminished NADH content, and a shift towards the green monomeric spectra after JC‐1 staining, indicating a reduced mitochondrial membrane potential. Surprisingly, NOR‐1 KD also reduced mTORC1 signaling, evidenced by reduced P70S6K and S6K phosphorylation.ConclusionsThese data suggest loss of NOR‐1 reduces oxidative gene expression, induces mitochondrial depolarization, and reduces NADH content, implicating NOR‐1 as an important regulator of energy homeostasis and muscle metabolic signaling. Investigations into the molecular hierarchy between NOR‐1 and PGC‐1α will delineate between primary and secondary effects of NOR‐1 deficiency.